title: Retinal expression of the X-linked juvenile retinoschisis (RS1) gene is controlled by an upstream CpG island and two opposing CRX-bound regions.
abstract: X-linked juvenile retinoschisis (XLRS) is an orphan retinal disease in males caused by mutations in the RS1 gene. Previously we have characterized cone-rod homeobox (CRX)-responsive elements in the promoter region of RS1 driving selective gene expression in the retina. Here, we expanded our identification and functional analysis of cis-regulatory elements controlling quantitative expression of RS1 in vitro and in vivo. Sequence analysis identified a CpG island 3kb upstream of the transcription start site (TSS). In addition, chromatin immunoprecipitation coupled to microarrays (ChIP-Chip) targeting the retinal transcription factor CRX was performed. Thereby, we identified a second CRX-bound region (CBR2) in the first intron of RS1 which contains six evolutionarily conserved CRX binding motifs. In vitro luciferase reporter gene assays and dsRed reporter electroporation of mouse retinal organ cultures demonstrated a strong constitutive and orientation-independent enhancing effect of the upstream CpG island. The intronic CBR2 potently suppressed CBR1-driven RS1 promoter activity in vitro but failed to regulate a CBR1-reporter in short-term cultured mouse retinae. We conclude that a CpG island enhancer and two CBRs may act in a combinatorial fashion to fine-tune RS1 transcript levels in the retina.
authors.affiliation: Institute of Human Genetics, University of Regensburg, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Kraus
authors.forename: Marcus
authors.initials: M
authors.lastname: Karlstetter
authors.forename: Yana
authors.initials: Y
authors.lastname: Walczak
authors.forename: Daniela
authors.initials: D
authors.lastname: Hilfinger
authors.forename: Thomas
authors.initials: T
authors.lastname: Langmann
authors.forename: Bernhard H F
authors.initials: BH
authors.lastname: Weber
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012160:Retina / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D046509:Tissue Culture Techniques; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D005136:Eye Proteins; D018398:Homeodomain Proteins; C491843:RS1 protein, mouse; D015534:Trans-Activators; C109288:cone rod homeobox protein; D049452:Green Fluorescent Proteins; D008156:Luciferases